U.S. patent application number 11/794146 was filed with the patent office on 2008-11-27 for mucoadhesive pharmaceutical compositions comprising chemoattractants.
This patent application is currently assigned to UNIVERSITE DE LIEGE. Invention is credited to Jacques Boniver, Luc Delattre, Philippe Delvenne, Brigitte Evard, Jean-Michel Foidart, Francis Frankenne, Ludivine Herman, Pascale Hubert, Agnes Noel.
Application Number | 20080293630 11/794146 |
Document ID | / |
Family ID | 34930173 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080293630 |
Kind Code |
A1 |
Herman; Ludivine ; et
al. |
November 27, 2008 |
Mucoadhesive Pharmaceutical Compositions Comprising
Chemoattractants
Abstract
The invention relates to a mucoadhesive pharmaceutical
composition comprising a polymer and a chemoattractant wherein the
pH of the composition is greater than 6 which is useful in the
treatment of a anogenital or oral disease, particularly an
anogenital or oral disease cause by the human papillomavirus.
Inventors: |
Herman; Ludivine; (Alleur,
BE) ; Hubert; Pascale; (Coutisse, BE) ;
Delvenne; Philippe; (Plainevaux, BE) ; Boniver;
Jacques; (Heusy, BE) ; Evard; Brigitte;
(Verlaine, BE) ; Delattre; Luc; (Hermee, BE)
; Frankenne; Francis; (Chaudfontaine, BE) ; Noel;
Agnes; (Durbuy, BE) ; Foidart; Jean-Michel;
(Trooz, BE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W., SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
UNIVERSITE DE LIEGE
Liege
BE
|
Family ID: |
34930173 |
Appl. No.: |
11/794146 |
Filed: |
December 12, 2005 |
PCT Filed: |
December 12, 2005 |
PCT NO: |
PCT/EP05/56709 |
371 Date: |
June 26, 2007 |
Current U.S.
Class: |
514/3.7 |
Current CPC
Class: |
A61K 47/38 20130101;
A61P 15/00 20180101; A61K 9/0031 20130101; A61K 47/34 20130101;
A61P 31/20 20180101; A61K 47/10 20130101; A61K 9/0034 20130101;
A61K 9/006 20130101; A61P 31/12 20180101; A61P 1/00 20180101; A61P
17/00 20180101; A61P 1/02 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61P 31/20 20060101 A61P031/20 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 2004 |
EP |
04106995.6 |
Claims
1. A mucoadhesive pharmaceutical composition comprising a polymer
and a chemoattractant wherein the pH of the composition is greater
than 6.
2. The composition as defined in claim 1 wherein the
chemoattractant is selected from the group consisting of
MIP3.alpha., H.beta.D2, MCP-1 and molgramostim.
3. The composition as defined in claim 1 which contains an
auxiliary chemoattractant which is selected from the group
consisting of granulocyte macrophage--colony recruiting factor
(GM-CSF) and HNP2 (human neutrophil defensin 2).
4. The composition as defined in claim 1 wherein the polymer is
selected from the group consisting of a natural gum, a
semi-synthetic material, and a synthetic material.
5. The composition as defined in claim 1 wherein the pH of the
composition is from 6 to 8, preferably the pH of the composition is
about 6.9.
6. The composition as defined in claim 1 for use in the treatment
of a squamous mucosa, preferably in the treatment of an anogenital
or oral disease, especially human papillomavirus.
7. The use of a composition as defined in claim 1 in the
manufacture of a medicament for use in the treatment of a squamous
mucosa, preferably in the treatment of an anogenital or oral
disease, especially human papillomavirus.
8. The method of treating an anogenital and/or oral disease which
method comprises administering a therapeutically effective amount
of a composition as defined in claim 1 to a patient in need of such
treatment.
Description
[0001] The present invention relates to a composition for the
treatment of a anogenital or oral disease, especially such a
disease caused by a human papillomavirus.
[0002] Human Papillomaviruses (HPV) are common sexually transmitted
pathogens inducing a spectrum of diseases ranging from benign
genital warts to invasive carcinomas. Some types of HPV have been
shown to be directly involved in the development of cervical cancer
and its precursors (squamous intraepithelial lesions (SIL)) (Bosch
et al, 1995). Squamous intraepithelial lesions refer to a group of
premalignant changes in the epithelium of the uterine cervix that
precede the onset of invasive cancer.
[0003] Additional environmental and/or host factors are probably
involved in malignant progression as suggested by the small number
of infected individuals developing cervical cancer and the
relatively long latency period before cancer emergence. The role of
the intrinsic immunity in controlling HPV infection and the
subsequent development of SIL is shown indirectly by the increased
frequency of HPV-associated lesions in patients with depressed
cell-mediated immunity (Petry et al, 1994; Ellerbrock et al, 2000).
The nature of an effective immune response to HPV infections is not
well understood, although cell-mediated immunity is thought to be
more important than humoral immunity (Thivolet et al, 1982; Wu et
al, 1994). Several studies have described a localized immune
dysfunction accompanying cervical HPV infections and associated
cervical lesions.
[0004] A way of improving the treatment of such conditions has been
sought.
[0005] According to the invention there is provided a mucoadhesive
pharmaceutical composition comprising a polymer and a
chemoattractant wherein the pH of the composition is greater than
6.
[0006] It has surprisingly been found that a chemoattractant
significantly enhanced the chemotaxis of an antigen presenting cell
and that the inclusion of a chemoattractant in a mucoadhesive
composition having a pH greater than 6 stimulated the infiltration
of an antigen presenting cell into a HPV-transformed epithelium.
Therefore, the composition according to the invention will be
useful in the treatment of a anogenital or oral disease,
particularly an anogenital or oral disease caused by the human
papillomavirus. A further useful feature of the invention is that
the chemoattractant did not interfere with the antigen presenting
ability of the antigen presenting cell.
[0007] A chemoattractant is generally understood to be a
pharmacological agent which modulates the recruitment of cells such
as a dendritic and/or Langerhans cell. The chemoattractant used in
the invention is preferably selected from the group consisting of
MIP3.alpha. (macrophage inflamatory proteine 3.alpha.), H.beta.D2
(human .beta. defensin 2), MCP-1 (monocyte chemotactic protein-1)
and molgramostim. Preferably two or more chemoattractants are used,
e.g. MIP3.alpha. and H.beta.D2.
[0008] The composition according to the invention preferably
contains an auxiliary chemoattractant which is selected from the
group consisting of granulocyte macrophage--colony recruiting
factor (GM-CSF) and HNP2 (human neutrophil defensin 2). A preferred
combination of chemoattractant and auxiliary chemoattractant for
use in the composition according to the invention is MCP-1 and
HNP2.
[0009] The composition according to the invention is preferably in
the form of a continuous structure, preferably having a solid-like
property. The composition is optionally either clear or opaque. In
the simplest composition according to the invention, the polymer is
a natural gum (e.g. xanthane), a semi-synthetic material (e.g.
methylcellulose, carboxymethyl-cellulose, hydroxyethyl-cellulose
hydroxypropylmethyl-cellulose or hydroxypropyl-cellulose) or a
synthetic material (e.g. a carbomer, polycarbophil and/or a
carboxyvinyl polymer).
[0010] The polymer is preferably an acrylic acid containing
polymer, more preferably it is a polycarbophil. The composition
according to the invention is preferably in the form of a
hydrogel.
[0011] The pH of the composition is preferably from 6 to 8. More
preferably it is about 6.9.
[0012] The composition according to the invention optionally
further comprises a preservative, buffer (especially an isotonic
buffer) and/or stabiliser such as methylparahydroxybenzoate,
parabens, EDTA, potassium sorbate and/or propylparahydroxybenzoate.
A suitable carrier, excipient and/or other agent may be included in
the composition according to the invention, e.g. to provide
improved transfer and/or delivery.
[0013] According to the invention there is also provided a
composition according to the invention for use in the treatment of
a squamous mucosa, preferably in the treatment of an anogenital or
oral disease, especially human papilloma virus.
[0014] By anogenital disease is meant vulvar, vaginal, cervical
and/or penile and/or anorectal disease.
[0015] According to the invention there is further provided use of
a composition according to the invention in the manufacture of a
medicament for use in the treatment of a squamous mucosa,
preferably in the treatment of an anogenital or oral disease,
especially human papillomavirus.
[0016] According to the invention there is also provided a method
of treating an anogenital and/or oral disease which method
comprises administering a therapeutically effective amount of a
composition according to the invention to a patient in need of such
treatment. Typically, treatment with direct administration is made
daily, weekly or monthly for a period of time sufficient to reduce,
prevent or ameliorate one or more symptoms.
[0017] For a typical composition having a volume of about 100 ml,
the amount of chemoattractant used in the composition is from long
to 500 .mu.g, preferably from 100 ng to 100 .mu.g, depending on the
chemoattractant used. A person of skill in the art will be able to
determine a suitable amount.
[0018] The invention is illustrated with reference to the Figures
of the accompanying drawings in which:
[0019] FIG. 1 is a graph showing chemotactic activity of different
molecules on DC wherein NC indicates non-conditioned medium; the
results are the mean.+-.SD of three experiments and asterisks
indicate statistically significant differences: **P<0.01;
[0020] FIG. 2 is a graph showing chemotactic activity of different
molecules on LC wherein NC indicates non-conditioned medium;
results are the mean.+-.SD of seven experiments and asterisks
indicate statistically significant differences: *P<0.05;
[0021] FIG. 3 shows the correlation between the penetration of DC
into organotypic cultures and the addition of exogenous
GM-CSF(C,D); MCP-1(E,F); and HNP2(G,H), shown by CD1a.sup.+
immunolabeled sections of organotypic culture of HPV.sup.+
keratinocytes wherein photographs A and B show DC infiltration in
the absence of chemotactic molecules and C-H show DC recruitment in
the presence of chemotactic molecules in liquid culture medium (C,
E, G) or included in the polycarbophil gel (D, F, H);
[0022] FIG. 4 shows the correlation between the penetration of LC
into organotypic cultures and the addition of exogenous GM-CSF
(C,D); MIP3.alpha. (E,F); and H.beta.D2 (G,H), shown by CD1a.sup.+
immunolabeled sections of organotypic culture of HPV.sup.+
keratinocytes wherein photographs A and B show DC infiltration in
the absence of chemotactic molecules and C-H show DC recruitment in
the presence of chemotactic molecules in liquid culture medium (C,
E, G) or included in the polycarbophil gel (D, F, H);
[0023] FIG. 5 shows quantitative evaluation of DC and LC
infiltration into organotypic cultures of HPV-transformed
keratinocytes wherein the penetration of DC (A) is tested in the
absence or in the presence of GM-CSF (used as control), MCP-1 and
HNP2 included or not in polycarbophil gel; and the infiltration of
LC (B) is evaluated in the absence or in the presence of GM-CSF,
MIP3.alpha. and H.beta.D2 included or not in the polycarbophil gel.
DC and LC were detected by immunolabeling with anti-CD1a antibody.
Results are expressed as infiltration depth in percentage of
epithelial sheet thickness (n=4 for each culture condition).
Asterisks indicate statistically significant differences:
*P<0.05; **P<0.01; ***P<0.001;
[0024] FIG. 6 shows that DC-mediated cytostatic activity is
maintained in the presence of chemokines and defensins. HPV.sup.+
keratinocytes were cultured in 96-well plates at 5.times.10.sup.3
cells/well. These cells were cultured with DC added at
4.times.10.sup.4 cell/well. After an incubation of 48 h in the
presence or in the absence of chemotactic molecules, cell
proliferation was measured by 3H-TdR incorporation. The results are
expressed as percentages of HPV.sup.+ keratinocyte proliferation in
the coculture compared to keratinocytes cultured alone, in the
presence or in the absence of chemokines or defensins. The results
are presented as means.+-.SD of 4 experiments using DC from
different donors; and
[0025] FIG. 7 shows that Chemokines and defensins did not alter the
ability of DC to stimulate a potent mixed lymphocyte reaction.
5.times.10.sup.4 DC irradiated with 2500 rads were cultured with
1.times.10.sup.5 PBMC in the presence of chemokines or defensins,
during 7 days. The cell proliferation was measured by 3H-TdR
incorporation. Proliferation index was obtained by comparing the
cell proliferation in the mixed DC-PBMC cultures added or not with
chemokines/defensins, with PBMC cultured alone with or without
chemokines/defensins. These results are presented as means.+-.SD of
3 experiments using DC from different donors.
[0026] The invention will now be illustrated with reference to the
following Examples which are not intended to limit the scope of the
invention defined herein.
EXAMPLE 1
[0027] A gel formulation according to the invention was prepared in
the usual manner from a therapeutically effective amount of MCP-1,
MIP3.alpha. or H.beta.D2, 3 wt. % hydroxyethylcellulose
(TYLOSE.RTM. H4000), 0.3 wt. % of potassium sorbate, 0.3 wt. % of
NaH.sub.2PO.sub.4 and purified water q.s. to 100 wt. %.
EXAMPLE 2
[0028] A gel formulation according to the invention suitable for
oral or anal delivery was prepared in the usual manner from a
therapeutically effective amount of MCP-1, MIP3.alpha. or H.beta.D2
with 7 g of hydroxypropylcellulose (Klucel GF) and an isotonic
buffer to 100 g. The isotonic buffer contains 1.36 g of
KH.sub.2PO.sub.4, 200 ml of purified water and 4N NaOH to give a pH
of 6.9.
EXAMPLE 3
[0029] A gel formulation according to the invention suitable for
oral or anal delivery was prepared in the usual manner from a
therapeutically effective amount of MCP-1, MIP3.alpha. or H.beta.D2
with 20 g of polyethylene polyoxypropylene block polymer (Lutrol
F127) and an isotonic buffer as defined in Example 2 to 100 g.
EXAMPLE 4
[0030] A gel formulation according to the invention suitable for
oral or anal delivery was prepared in the usual manner from a
therapeutically effective amount of MCP-1, MIP3.alpha. or H.beta.D2
with 1.5 g of polyacrylic acid (Carbopol 974P), 0.5 g of EDTA,
trometamol to a pH of 6.9 and purified water to 100 g.
EXAMPLE 5
[0031] A gel formulation according to the invention suitable for
oral or anal delivery was prepared in the usual manner from a
therapeutically effective amount of MCP-1, MIP3.alpha. or H.beta.D2
with:
TABLE-US-00001 Hydroxypropylcellulose (Klucel GF) 7 g Parabens q.s.
Isotonic buffer (as in Example 2) to 100 g
EXAMPLE 6
[0032] A gel formulation according to the invention suitable for
oral or anal delivery was prepared in the usual manner from a
therapeutically effective amount of MCP-1, MIP3.alpha. or H.beta.D2
with:
TABLE-US-00002 Polyethylene polyoxypropylene 20 g block polymer
(Lutrol F127) Parabens q.s. Isotonic buffer (as in Example 2) to
100 g
EXAMPLE 7
[0033] A gel formulation according to the invention suitable for
oral or anal delivery was prepared in the usual manner from a
therapeutically effective amount of MCP-1, MIP3.alpha. or H.beta.D2
with:
TABLE-US-00003 Polyacrylic acid (Carbopol 974P) 1.5 g EDTA 0.5
Parabens q.s. Purified water to 100 g Trometamol to pH 6.9
EXAMPLE 8
Materials and Methods
Culture of Cervical HPV-Transformed Keratinocyte Cell Lines
[0034] SiHa and CasKi are tumorigenic cervical carcinoma-derived
keratinocyte cell lines (Friedl et al, 1970; Pater et al, 1985;
Auersperg et al, 1962). SiHa cell line contains one copy and CasKi
contains approximately 600 copies of integrated HPV-16 DNA. These
HPV-transformed keratinocyte cell lines were cultured in growth
medium composed of 1/3 mixture of HAM F12 (GIBCO BRL, Nord Island,
N.Y., USA)/Dulbecco's modified Eagle's medium (GIBCO BRL),
supplemented with 0.4 .mu.g/ml hydrocortisone (Sigma Chemical Co.,
St Louis, Mo.), 2 ng/ml epidermal growth factor (Sigma), 10% fetal
calf serum (Life Sciences International, Zelik, Belgium), 2 mmol/L
L-glutamine (GIBCO BRL), 10 mmol/L Hepes (GIBCO BRL), 1 .mu.g/ml
fungizone (GIBCO BRL), 1 mmol/L sodium pyruvate (GIBCO BRL), 3000
U/ml penicillin-streptomycin (GIBCO BRL), 10.sup.-10 mol/L cholera
toxin (Sigma), 5 .mu.g/ml insulin (Sigma), 20 .mu.g/ml adenine
(Sigma), 5 .mu.g/ml human transferrin (Sigma), and 15.10.sup.-4
.mu.g/ml 3,3',5-triiodo-L-thyronine (Sigma).
Dendritic Cell Cultures
[0035] Dendritic cells (DC) were generated by culturing adherent
fraction of human PBMC as previously described (Hubert et al, 1998;
Sallusto and Lanzavecchia, 1994). Briefly, Peripheral Blood
Mononuclear Cells (PBMC) were isolated from leukocyte-enriched
buffy-coats by centrifugation on Ficoll-Hypaque. After washings at
low centrifugation speed to discard a maximum of platelets, PBMC
were plated in six-well dishes (Nunclon Multidishes, NUNC, Denmark)
at a density of 10.times.10.sup.6 PBMC per well in 3 ml of medium
without FCS. After 18 h at 37.degree. C., non-adherent cells were
discarded by two gentle washes and the plastic adherent fraction
was cultured with 800 U/ml human recombinant GM-CSF (Amoytop
Biotech, Xiamen, China) and 40 U/ml IL4 (ImmunoTools, Friesoythe,
Germany) in 3 ml of RPMI 10% FCS-50 .lamda.M Mercaptoethanol.
Cultures were fed every 3 days with fresh medium containing
cytokines and harvested with PBS-EDTA 1 mM at day 7.
Langerhans Cell Cultures
[0036] Cord blood mononuclear cells (CB MNC) were recovered after
discontinuous density gradient centrifugation using Lymphopre p
(Nycomed Pharma As, Oslo, Norway) within 24 hours after collection.
CD34.sup.+ cells were isolated from CB MNC using the MACS Direct
CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec GmBH, Bergisch
Gladbach, Germany) and MiniMACS separation columns (Miltenyi
Biotec) according to the manufacturer's protocol. Fifteen 10.sup.4
CD34.sup.+ cells were seeded in T25 flasks (Sarstedt, Inc, Newton,
N.C.) in 10 ml of RPMI 1640 medium supplemented with 10% FCS,
antibiotics and 50 .mu.M mercaptoethanol (all from GIBCO-BRL).
Cultures were supplemented with previously optimized concentrations
of the following human molecules: SCF (20 ng/ml, specific activity
(SA)>5 10.sup.5 U/mg), TPO (10 U/ml, SA >1 10.sup.6 U/mg),
F1T3-L (25 ng/ml, SA >2 10.sup.5 U/mg), GM-CSF (200 U/ml, SA:
11, 1 10.sup.6 U/mg), TNF.alpha. (50 U/ml, SA >2 10.sup.7 U/mg),
IL4 (100 U/ml, SA >2 10.sup.6 U/ml) and TGF-.beta.1 (5 ng/ml or
12.5 ng/ml, SA >2 10.sup.8 U/mg). All these agents were
purchased from PeproTech (Rockey Hill, N.J.), except for GM-CSF and
IL4 which were obtained from Amoytop (Amoytop Biotech, Xiamen,
China) and Biosource (Nivelles, Belgium), respectively. The cells
were cultured at 37.degree. C. in a humidified atmosphere and in
the presence of 5% CO.sub.2. At day 7, the cellular density was
adjusted to 2.10.sup.4 per cm.sup.2 and the cells were fed, at days
7 and 14, with GM-CSF, IL4, TNF.alpha. and TGF-.beta.1 at the same
concentrations as those used at the start of the culture, except
for TGF-.beta.1 at day 14 which was 12.5 ng/ml for all the
cultures. At day 18, cells were collected from cultures by vigorous
pipetting to prepare single cell populations (Hubert et al, in
press).
Chemotaxis Assay
[0037] Cell migration was evaluated using a chemotaxis microchamber
technique (48-well Boyden microchamber; Neuroprobe, Cabin John,
Md., USA). The lower wells of the chemotaxis chamber was filled
with 27 of non conditioned medium, human fibroblasts derived
cell-conditioned medium or the differents chemokines studied.
Nonconditioned medium was used as control for random migration.
Each condition was repeated six times. After phenotypic
characterisation, DC were harvested and 55 .mu.l of DC suspension
(2.times.10.sup.6 cells/ml) were applied to the upper wells of the
chamber, with a polyvinylpyrollidone-free polycarbonate membrane
8-.mu.m pore filter (Poretics Corp., Livermore, Calif.) separating
the lower wells. These membranes were coated by incubation with 100
.mu.g/ml gelatin in 0.1% acetic acid solution. The chamber was
incubated for 5 h at 37.degree. C. in a 5% CO.sub.2/95% air
atmosphere. The cells having migrated to the underside of the
filter were fixed and stained with Diff Quick Stain set (Baxter
Diagnostics AG, Dudingen, Switzerland). The upper side of the
filter was scraped to remove residual non migrating cells. One
random field was counted per well using an eyepiece with a
calibrated grid to evaluate the number of fully migrated cells.
[0038] The same tests were realized for LC using standard 5-.mu.m
pore polyvinylpyrollidone-free polycarbonate filters to separate
the upper and the lower wells.
Preparation of the Hydrogel
[0039] Polycarbophil (Noveon AA1) was supplied from Noveon
(Brussels, Belgium). The polycarbophil gel (1.5% w/w) was prepared
by dispersing the Noveon AA1 in keratinocyte growth medium. The
mixture was stirred until thickening occurred and then neutralized
by dropwise addition of 40% (w/w) trometamol, until a transparent
gel appeared. The quantity of trometamol was adjusted to achieve pH
7.
Organotypic Cultures
[0040] Organotypic cultures of HPV-transformed keratinocyte cell
lines were prepared by procedures slightly modified from those
described previously (Delvenne et al, 1995; Merrick et al, 1992).
For the preparation of dermal equivalents, a collagen matrix
solution was made with 32 mg of collagen (cellagen solution AC-5,
type I, ICN, Biomedical, Asse-Relegen, Belgium) mixed on ice with
1.6 ml of 0.1% acetic acid, 1 ml of chilled 10-fold concentrated
Hanks' buffer supplemented with phenol red and 1N NaOH to give a pH
of 7.2. One milliliter of FCS containing 5.times.10.sup.5 normal
human fibroblasts was then added. One milliliter of
collagen/fibroblast solution was poured into 24-well plates
(Nunclon Multidishes, Nunc, Roskilde, Denmark) and allowed to
solidify at 37.degree. C. for 1 hour. The final concentrations of
collagen and fibroblasts were 3.2 mg/ml and 5.times.10.sup.4
cells/ml, respectively. After gel equilibration with 1 ml of growth
medium overnight at 37.degree. C., 30.10.sup.4 HPV-transformed
keratinocytes resuspended in 50 .mu.l of growth medium were seeded
on top of the gels and maintained submerged for 48 hours. The
collagen rafts were raised in 25-mm tissue culture insert (8-.mu.m
pore size; Nunc) and placed onto stainless-steel grids at the
interface between air and liquid culture medium. Epithelial cells
were then allowed to stratify over 20 days. After stratification of
keratinocytes, DC were seeded on top of the in vitro-formed
epithelium at a concentration of 4.10.sup.5 cells/50 .mu.l of
keratinocyte growth medium. The liquid culture medium or the
polycarbophil gel was supplemented or not with the different
chemokines/defensins at the following concentrations: 500 ng/ml of
Monocyte Chemotactic Protein 1 (MCP-1, PeproTech, Rocky Hill,
N.J.), 750 ng/ml of Human Neutrophil Peptide-2 (HNP2,
Sigma-Aldrich, St Louis, Mo.), 750 ng/ml of Human .beta. Defensin-2
(H.beta.D2, PeproTech, Rocky Hill, N.J.) or 500 ng/ml of Macrophage
Inflammatory Protein 3.alpha. (MIP3.alpha., PeproTech, Rocky Hill,
N.J.). After 48 hours at 37.degree. C., the collagen rafts were
harvested. The cultures were then embedded in OCT compound (Tissue
Tek, Sakura, The Netherlands) at -70.degree. C. and sectioned with
a cryostat microtome for the immunohistochemical analysis.
Immunohistochemistry
[0041] The density of DC migrating into the epithelial layer was
assessed by the avidin-biotin-peroxidase technique (Vectastain ABC
kit, Vector Laboratories, Burlingame, Calif.) with an anti-CD1a
monoclonal antibody (clone NA1/34 from Dako, Glostrup, Denmark).
Eight-micron frozen sections were fixed in cold acetone for 3
minutes, and endogenous peroxidases were blocked with 0.1%
H.sub.2O.sub.2 for 30 minutes. Sections were then incubated
sequentially with anti-CD1a antibody (at a 1/40 dilution in PBS
containing 2% bovine serum albumin (BSA)) for 1 hour, with a
biotinylated mouse anti-Ig antibody for 30 minutes, and with
streptavidin/horseradish peroxidase/avidin/biotin complex for
another 30 minutes. Positive cells were visualized by a
3,3'-diaminobenzidine substrate (DAB). The sections were
counterstained with hematoxylin.
Assessment of CD1a+ Cell Infiltration in Organotypic Cultures.
[0042] The DC/LC infiltration in the organotypic cultures was
evaluated by measuring the distance between the top of the
epithelium and individual DC with a computerized system of image
analysis (CAS, Becton Dikinson, Erembodegem, Belgium). The ratio
between the infiltration depth and the thickness of the culture was
then calculated. The percentage ratio was 0% for DC/LC staying on
the top of the culture and 100% for DC/LC reaching the bottom of
the epithelium.
Keratinocyte Growth Inhibition Assay
[0043] HPV-transformed keratinocyte cell lines (5.times.10.sup.3
cells/well) with or without DC (40000 DC/well) were cultured in
96-well plates (Nunclon Surface, NUNC) with or without
chemokines/defensins. Proliferation was measured after 48 h at
37.degree. C., following an 18 h incubation with 0.4 .mu.Ci/well of
[.sup.3H]thymidine (6.7 Ci/mmol, Moravek Biochemicals, Brea,
Calif.). [.sup.3H]thymidine can incorporate DNA during the S phase
of dividing cell cycle. DNA was harvested by an automated sample
harvester (Packar, Can berra, Tilburg, The Netherlands) and
thymidine incorporation was analysed by using a liquid
scintillation counter (Top Count, Packard, Can berra). The data are
presented as the percentage of proliferation calculated by using
the following formula:
% proliferation=(test cpm/control cpm).times.100
where test cpm is [.sup.3H]thymidine incorporation by keratinocytes
cultured with DC in the presence or not of chemokines/defensins and
control cpm is [.sup.3H]thymidine incorporation by keratinocytes
without DC but with or without chemokines/defensins.
Mixed Lymphocyte Reaction Assay
[0044] Stimulator populations (dendritic cells) were harvested and
irradiated with 2500 rads. After a wash and a centrifugation, the
cells were adjusted to 50000 DC/100 .mu.l in RPMI-1640 medium
containing 5% human pooled AB serum. Stimulator cells (50000
DC/well) were then added to round-bottomed 96-well plates (Nunclon)
containing 1.times.10.sup.5 allogeneic PBMC per well and the
differents chemokines/defensins. The tests were performed in
quadruplate. A proliferative response was measured after 7 days of
culture by adding 0.4 .mu.Ci [.sup.3H]thymidine (6.7 Ci/mmol,
Moravek Biochemicals, Brea, Calif.) to each well. DNA was harvested
18 h later by an automated sample harvester (Packar, Can berra,
Tilburg, The Netherlands) and counted in a liquid scintillation
counter (Top Count, Packard, Can berra).
Statistical Analysis
[0045] Statistical analysis was performed by using the Unpaired
Student t test (Instat Mac 2.01 software; Graph-Pad Software, San
Diego, Calif.). Differences were considered statistically
significant when P<0.05.
Results
HNP2 and MCP-1 Induce the Chemotaxis of DC
[0046] To identify molecules that could influence the migration of
DC, we conducted a migration assay using a Boyden microchemotaxis
chamber. DC generated for this study were judged to be 90% pure
based on several criteria, including morphology, forward-scatter
and side-scatter values by flow cytometry, and surface phenotype
(data not shown).
[0047] As shown in FIG. 1, the addition of GM-CSF (1 ng/ml) known
to be chemoattractant for DC (Hubert et al, 1999) significantly
enhanced the chemotaxis of DC, compared with the nonconditioned
medium used to assess the chemokinesis. Interestingly, MCP-1 and
HNP2 increased the migration of DC at respectively 500 ng/ml and
750 ng/ml which were previously determined in a preliminary work to
be the optimal concentrations. Besides, the chemotactic activity of
MCP-1 and HNP2 was similar to the level of chemotaxis induced by
GM-CSF.
H.beta.D2 and MIP 3.alpha. Induce the Chemotaxis of LC
[0048] In order to determine whether LC could be attracted by
chemotactic molecules, Boyden chamber assay was performed with in
vitro generated LC. LC were generated from CD34.sup.+ cord blood
progenitors cultivated in the presence of hematopoietic growth
factors (TPO, SCF, Flt3L), cytokines (GM-CSF, TNF.alpha., IL4) and
TGF-.beta.1 (Hubert et al, in press). They exhibited morphological,
immunohistochemical (CD1a.sup.+, CD207.sup.+, E-cadherin.sup.+,
CLA.sup.+ and CCR6.sup.+) and ultrastructural features (Birbeck
granules) of LC as verified by FACS analysis and electron
microscopy (data not shown).
[0049] As observed for DC, GM-CSF (10 ng/ml) induced a
significantly higher LC migration than the nonconditioned medium.
The mobility of LC in the presence of MIP3.alpha..sub.-- 500 ng/ml)
or H.beta.D2 (750 ng/ml) was identical to that observed with GM-CSF
(FIG. 2).
Chemokines and Defensins, Included in a Polycarbophil Gel Stimulate
the Infiltration of DC/LC into Organotypic Cultures of
HPV-Transformed Keratinocytes.
[0050] It was investigated whether the addition of MIP3.alpha. and
H.beta.D-2 or MCP-1 and HNP2 could modulate the ability of LC and
DC respectively to infiltrate an in vitro-formed (pre)neoplastic
epithelium, reminiscent of a cervical high-grade lesion observed in
vivo. After 20 days of culture, the HPV-transformed keratinocyte
cell lines grown on a collagen gel at the air/liquid interface,
produced an epithelial layer of up to 10-15 cells in thickness.
These cells appeared disorganized and highly atypical throughout
the full thickness of the epithelium as observed in high-grade
lesion biopsies.
[0051] DC or LC were layered on the top of these cultures in the
presence or in the absence of chemoattractant molecules. In order
to evaluate the possibility to topically apply these molecules in
an immunotherapeutic approach, chemokines and defensins were
included in a polycarbophil gel. The ability of chemotactic
molecules to influence the migration of DC/LC was determined by
evaluating the density of CD1a.sup.+ cells in sections of
organotypic cultures at 48 hours following chemoattractant
addition. FIG. 3 illustrates representative experiments showing
CD1a-labeled DC in HPV-transformed organotypic cultures incubated
or not with chemotactic molecules included or not in polycarbophil
gel. FIG. 4 represents similar experiments performed with LC.
[0052] In HPV-transformed keratinocyte organotypic cultures, LC/DC
poorly infiltrated the epithelial layer in the absence of
chemoattractant (FIG. 3, A-B and FIG. 4, A-B). In contrast,
addition of GM-CSF, the positive control, caused a significant
increase in the density of LC/DC observed in the epithelial layer
(FIG. 3, C-D and FIG. 4, C-D). The addition of chemokines and
defensins in growth medium induced a recruitment of DC/LC similar
to that observed with GM-CSF (FIG. 3 C,E,G and FIG. 4 C,E,G).
Interestingly, this infiltration was also present when the
chemoattractants were included in the polycarbophil gel (FIG. 3 D,
F, H and FIG. 4 D, F, H).
[0053] Quantitative analysis of DC/LC infiltration was performed by
evaluating the infiltration depth of all CD1a.sup.+ cells
throughout the full thickness of organotypic cultures (FIG. 5).
[0054] When the medium of organotypic culture was supplemented with
MCP1 or HNP2, the infiltration of DC increased compared to the
basal infiltration without chemoattractant, and reached an
infiltration level equivalent to that obtained with GM-CSF (FIG. 5,
A). The inclusion of chemoattractant molecules in the polycarbophil
gel did not modify the DC infiltration compared with that observed
in the presence of chemoattractants in the culture medium.
[0055] The infiltration of LC was also increased in the presence of
MIP3.alpha. or H.beta.D-2 (FIG. 5, B). When the medium of
organotypic cultures was supplemented with H.beta.D-2, the
migration of LC was slightly lower compared with that observed in
the presence of GM-CSF. The addition of MIP3.alpha._ induced a
recruitment similar to that obtained with GM-CSF. The
chemoattractants included in liquid culture medium or in
polycarbophil gel recruited LC in a similar manner.
[0056] To determine the potential influence of chemokines/defensins
on DC/LC differentiation, we performed a double-immunostaining
(CD1a/CD14) of organotypic cultures. The phenotype of DC/LC
infiltrating organotypic cultures in the presence of
chemokines/defensins was not altered compared with that established
before the migration (data not shown).
The Cytostatic Activity of DC Against HPV+ Keratinocytes is not
Modified in the Presence of Chemokines/Defensins.
[0057] Since DC are able to inhibit the growth of HPV-transformed
keratinocytes (Hubert et al, 2001) in vitro, we wanted to determine
the influence of chemotactic molecules on this cytostatic activity.
We performed a 48 h growth-inhibition assay by coculturing
HPV.sup.+ keratinocytes with DC in the presence or not of different
chemoattractants.
[0058] DC exhibited a significant growth inhibition effect on
HPV.sup.+ keratinocytes since DC reduced the proliferation of
HPV.sup.+ keratinocytes to 56% compared to cultures without DC.
Neither defensins nor chemokines modified the cytostatic activity
of DC against HPV.sup.+ keratinocytes.
Chemokines and Defensins do not Affect the Antigen Presentation
Function of DC in a Mixed Lymphocyte Reaction (MLR)
[0059] The lymphocyte proliferation was not changed in the presence
of chemokines or defensins only (data not shown).
[0060] As shown in FIG. 7, DC in the presence or in the absence of
chemotactic molecules did not differ significantly in their ability
to stimulate the T lymphocyte proliferation. Consequently, the
capacity of DC to present antigen to T lymphocytes was not
affected.
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